Genetic modiﬁcation of amino acid metabolismin woody plants
Departamento de Biología Molecular y Bioquímica,Facultad de Ciencias e Instituto Andaluz de Biotecnología,Unidad Asociada UMA-CSIC,Campus
de Teatinos,Universidad de Málaga,29071 Malaga,Spain
Department of Biological Sciences,Rutgers University,University Heights,Newark,NJ 07102,USA
Received 18 November 2002;accepted 13 January 2003
Forest trees comprise a large group of angiosperm and gymnosperm species of economic importance that play a crucial role in the
ecosystems.Nitrogen is frequently a limiting factor for growth of forest trees,thus development of a fundamental understanding of nitrogen
assimilation and metabolism is particularly important in broadening our understanding of fundamental tree biology.There are a number of
fundamental ways in which woody plants differ from herbaceous species,including seed dormancy and germination,growth habit and
enhanced secondary development,management of reduced nitrogen during dormancy,and the metabolic requirements for secondary growth,
a major sink for both reduced nitrogen and carbon.Poplar species (Populus spp.) have emerged as model systems for research in woody
angiosperms.Modiﬁcation of metabolism using genetic engineering approaches has recently focussed on altering the biosynthesis of
glutamine,polyamines,glutathione,and lignin.These approaches potentially affect plant development and stress tolerance.The aim of this
minireviewis to integrate the experimental genetic engineering approaches in the context of developing an increased understanding of overall
nitrogen and amino acid metabolismin trees.
©2003 Éditions scientiﬁques et médicales Elsevier SAS.All rights reserved.
Keywords:Nitrogen assimilation;Plant metabolism;Trees;Transgenic plants
Progress in nitrogen assimilation and amino acid biosyn-
thesis is of special interest for plant scientists and breeders,
since plant development and biomass production depend on
the availability of inorganic nitrogen in soil,and nitrogen
fertilizers are widely used to get better yields.In addition,
key enzymes in amino acid biosynthesis are the target of a
number of relevant herbicides that are used for weed control,
and the synthesis of amino acids is also associated to resis-
tance to stress in plants .Therefore,an increased knowl-
edge on nitrogen metabolism and the generation of plants
with higher efﬁciency in nitrogen assimilation and metabo-
lism are of broad interest for plant breeders,especially to
decrease the cost/production ratio,that includes relevant is-
sues such as (a) increase in yield,(b) decrease in the pollution
associated to the use of fertilizers,(c) improving the resis-
tance of cultures to different stresses and (d) the control of
competitive and undesirable weeds.
Most of the studies in nitrogen metabolism have been
performed in annual herbaceous plants,and much less is
known on this subject in woody perennial species.The aimof
this minireviewis to provide an overviewof the recent works
on genetic manipulation of amino acid metabolismin woody
plants,including ammoniumassimilation and glutamine bio-
synthesis,polyamine metabolism,and glutathione biosyn-
thesis.Special attention will focus on the integration of the
experimental approaches in the context of overall nitrogen
and amino acid metabolismin trees (Fig.1).Arelated meta-
bolic pathway is the biosynthesis of the aromatic amino acids
c-glutamylcysteine synthetase;GOGAT,glutamate synthase;GS,glutamine
synthetase;GSHs,glutathione synthetase;QTLs,quantitative trait loci;
ODC,ornithine decarboxylase;Put,putrescine;VSPs,vegetative storage
* Corresponding author.
E-mail address:email@example.com (F.M.Cánovas).
Present address:Department of Plant,Soil and Entomological Scien-
ces,University of Idaho,College of Agriculture,Research and Extension
Center,P.O.Box 870,Aberdeen,ID 83210,USA.
Plant Physiology and Biochemistry 41 (2003) 587–594
©2003 Éditions scientiﬁques et médicales Elsevier SAS.All rights reserved.
phenylalanine and tyrosine (through the shikimate pathway),
which leads to the biosynthesis of lignin.Reviews addressing
lignin manipulation and wood formation have recently ap-
peared [1,3,26] and,therefore,this topic will not be consid-
ered in this paper.
2.Trees as experimental plant models,relevance
Most of our knowledge on plant metabolismand physiol-
ogy has been gained using cell cultures,seedlings or whole
plants of herbaceous species,including model systems and
other species of agronomic importance.Recently,it has been
shown that a number of trees may be suitable for both basic
and applied research [32,63].Progress from molecular and
physiological studies in trees has been constrained by various
difﬁculties that trees present for experimental work,includ-
ing their large physical size,the usually large genome in
many species of economic interest,long life cycle of peren-
nial species,difﬁculties for genetic transformation and re-
generation in vitro,and difﬁculties for molecular and bio-
chemical analyses.However,there are important reasons to
study woody plants.They provide a range of products of
commercial interest,including wood,pulp,wood products,
and important secondary metabolites.Trees are of great envi-
ronmental interest for reforestation programs,soil retention,
as essential components of the natural landscape,and for
recreation.Moreover,forest ecosystems play a crucial role in
global carbon budgeting,responses to global climate change,
and maintenance of biodiversity.
Signiﬁcant research efforts have focussed on the develop-
ment of protocols for mass clonal propagation of forest
species of economic importance,including a number of coni-
fers (such as pines,spruces and Douglas ﬁr) and angiosperm
species of the genera Eucalyptus and Populus .Genetic
transformation of conifers,the most important group of gym-
nosperms,is still a difﬁcult task,although protocols for
Agrobacterium-mediated transformation have been reported
for embryogenic cultures of spruce and loblolly pine .
Furthermore,the transformation of shoots of loblolly pine
using Agrobacterium inﬁltration has also been recently re-
ported .Somatic embryogenesis represents a useful
model to study the developmental regulation of gene expres-
sion .Such recent technological advances are opening
the possibility of modifying the metabolism in gymno-
In angiosperm forest trees,Populus has emerged as the
model for experimental research [9,56].The poplar species
have small genomes and are characterized by an easy vegeta-
tive propagation.Furthermore,some genotypes are amenable
to transformation via A.tumefaciens.In addition,the avail-
ability of fast growing poplar clones allowfor the transfer of
molecular studies to ﬁeld trials in a relatively short period of
time .Moreover,because of their distinct juvenile and
mature phases of growth,woody plants offer opportunities to
study speciﬁc metabolic processes,thus,complementing our
knowledge acquired fromherbaceous plants.
3.Modiﬁcation of nitrogen metabolism in trees
3.1.Genetic manipulation of glutamine biosynthesis
Availability of inorganic nitrogen in the soil is frequently a
limiting factor for plant growth.Thus,the uptake of inorganic
nitrogen and its incorporation to amino acids have been the
main area of interest in plant biochemistry and physiology.
Knowledge on nitrogen assimilation has been recently re-
vised,and research efforts have centered on elucidation of
key steps of the process,including nitrate uptake and reduc-
tion,incorporation of ammonium into glutamine,and bio-
synthesis of glutamate .Although the physiology of
nitrogen uptake is well characterized,the modiﬁcation of
nitrate uptake using transgenic approaches is complicated,
because of the existence of multiple genes involved in nitro-
gen transport systems in plants [25,45].However,the reduc-
tion of nitrate to ammonium and the assimilation of ammo-
nium into glutamine are well understood.Thus,by the
introduction of speciﬁc transgenes,transgenic plants could
provide a means to study the role of various factors that affect
and regulate ammoniumassimilation.
Early studies in the 1990s indicated that an increase in the
level of key enzymes,including nitrate and nitrite reductases,
glutamine synthetase (GS) and asparagine synthetase,in
transgenic plants resulted in very limited or no effect on the
phenotype of the modiﬁed plant [18,57].Therefore,co-
transformation with other enzyme(s) to avoid the generation
of new limiting steps in the metabolic pathways was sug-
gested .Nearly one decade of research has been neces-
An overview of nitrogen metabolism depicting the major pathways
studies in woody plants.ADC,arginine decarboxylase;c-ECS,
c-glutamylcysteine synthetase;GOGAT,glutamate synthase;GS,glutamine
synthetase;GSHs,glutathione synthetase;ODC,ornithine decarboxylase;
588 F.Gallardo et al./Plant Physiology and Biochemistry 41 (2003) 587–594
sary to demonstrate that the modiﬁcation of GS alone may
affect the biosynthesis of nitrogen compounds and plant
development.GS exists as two different isoenzymes in most
plant tissues:a chloroplastic enzyme (GS2),which is in-
volved in the assimilation of ammoniumfromnitrate reduc-
tion and photorespiration in photosynthetic cells;a cytosolic
enzyme (GS1),which is expressed mainly in roots and non-
photosynthetic tissues.The biological role of GS1 is not well
deﬁned,but it seems to be involved in the primary assimila-
tion of ammonium from the soil and in the assimilation of
ammoniumreleased through other secondary metabolic pro-
cesses other than photorespiration.Since GS1 in the leaf is
expressed speciﬁcally in the vascular bundles,a role for GS1
in the transport of glutamine to other plant organs has been
considered [13,47].However,recent work fromour group on
GS expression in pine seedlings has demonstrated the pres-
ence of GS1 in photosynthetic cells growing vegetatively
,and in the mesophyll cells of tomato leaves infected by
Pseudomonas syringae .
Using the transformation approaches,modiﬁcation of the
expressions of GS1 and GS2 has been achieved in a number
of species including tobacco,Lotus,rice,and wheat,and in
the woody perennial poplar.Results conﬁrmthe relevance of
GS isoenzymes in plant development,biomass production,
and yield (reviewed in ).In angiospermand gymnosperm
trees,the study and the modiﬁcation of nitrogen assimilation
hold special interest.During the early stages of development,
the GS expression is regulated somewhat differently in coni-
fers than in angiosperms,speciﬁcally affecting the biogen-
esis of chloroplasts.In conifers,the regulation of develop-
ment of chloroplasts and the expression of chloroplast-
speciﬁc enzymes are less dependent on light than in
angiosperm species.This includes the expression of genes
implicated in ammoniumassimilation [12,55].The economy
of reduced nitrogen is of special importance in trees,since
inorganic nitrogen is incorporated into amino acids,and to a
great extent excess nitrogen is accumulated in the bark of the
stem as vegetative storage proteins (VSPs).Moreover,
woody tissues of the trunk of the trees are important sinks for
the carbon and nitrogen assimilated during the tree life cycle.
The demand for human use of products derived from forest
trees,for example wood,is increasing.Knowledge on meta-
bolic and developmental processes relating to wood and
biomass production to efﬁciency of nitrogen utilization will
lead to applications in increasing overall forest productivity.
The mechanisms whereby trees manage the reduced nitro-
gen,during the onset of dormancy and resumption of active
growth,are of importance in developing a complete under-
standing of nitrogen homeostasis.VSPs serve as sinks for
re-absorption of nitrogen from senescing leaves;thus,they
act as a reservoir of reduced nitrogen to support growth
during the start of each growing season .Recent research
has centered on the regulation of expression of VSPs and the
effects of nitrogen status,photoperiod,and abiotic stress
.Changes in growth in response to environmental factors
may alter carbon and nitrogen partitioning and,thus,provoke
VSP production .Accumulation of VSPs appears to be a
component of the overall nutrient use efﬁciency of the plant.
Thus,manipulation of VSP production could offer an impor-
tant avenue for enhancing biomass production in trees.
Nitrogen assimilation and mobilization are crucial pro-
cesses for the growth and development of perennial species.
Modiﬁcation of the expression of key enzymes in nitrogen
metabolismis a reasonable strategy for enhancing growth of
forest trees.Increased levels of GS have been achieved in
transgenic poplar by the ectopic constitutive expression of a
cytosolic pine GS1,under the direction of a CaMV 35S
promoter  (Table 1).The introduced pine GS is a cytoso-
lic enzyme,and its ectopic expression in poplar has resulted
in the production of a pine holoenzyme in photosynthetic
cells .Although the role of cytosolic GS is not well
deﬁned in the leaves of angiosperms,its expression in pho-
tosynthetic tissues has been associated with responses to
biotic and abiotic stresses,fruit ripening,and leaf senescence
[5,48,10,23].Therefore,the modiﬁcation of GS1 levels may
have an important effect not only on nitrogen partitioning,
but also on stress tolerance.Although there are several re-
ports of transgenic herbaceous plants with altered chloro-
plastic GS contents ,the discussion here will focus on an
alteration of cytosolic GS expression.
The analysis of transgenic poplar lines expressing the pine
cytosolic GS has revealed that the ectopic expression of GS1
in the leaf leads not only to increased GS activity,but also to
enhanced chlorophyll and protein contents [20,22].More-
over,the enhancement of vegetative growth with respect to
untransformed plants (Fig.2) has been reported.Similar
results have been obtained in herbaceous plants [21,41],
indicating that the modiﬁcation of cytosolic GS levels may
be an appropriate approach for improving growth of crop
species.Interestingly,the overexpression of GS1 in Lotus
corniculatus,a legume plant,resulted in premature ﬂowering
and early senescence .This apparent acceleration of
development is an interesting observation and may have
application in accelerating ﬂowering in plants with long
juvenile periods,as in the case of woody perennials.
Greenhouse studies of transgenic poplars overexpressing
pine GS1 showed that enhanced GS activity in young leaves
was correlated with increases in height growth [20,22].Fu-
Genetic manipulation of glutamine,polyamine and glutathione biosynthesis in woody plants
Host species Transgene Gene source Promoter Gene transfer Reference
P.tremula ×P.alba Glutamine synthetase Pinus sylvestris 35S (CaMV) Agrobacterium tumefaciens [20,22]
P.nigra ×P.maximowiczii Ornithine decarboxylase Mus musculus 35S (CaMV) Biolistic bombardement [6,7]
P.tremula ×P.alba c-Glutamylcysteine synthetase Escherichia coli 35S (CaMV) Agrobacterium tumefaciens [42,43]
P.tremula ×P.alba Glutathione synthetase Escherichia coli 35S (CaMV) Agrobacterium tumefaciens [19,42]
589F.Gallardo et al./Plant Physiology and Biochemistry 41 (2003) 587–594
entes et al. reported that a higher performance of trans-
genic tobacco plants overexpressing GS1 was observed,spe-
ciﬁcally when inorganic nitrogen availability was low.
Similar results have been observed for GS-overexpressing
poplars grown under low(0.3 mM) and high (10 mM) nitrate
regimes (Man and Kirby,unpublished data).Similar ﬁndings
have also been reported by Oliveira et al. using trans-
genic tobacco plants.Recent work using
shown that transgenic poplars have enhanced nitrogen as-
similation efﬁciencies,particularly under conditions of low
nitrate availability (Man and Kirby,unpublished data).These
results suggest that GS-overexpressing poplars may be better
able to exploit nitrogen resources in the soil and,thus,require
lower nitrogen fertilization regimes.This could result in
reduced risk of pollution,as posed by current agricultural
Transgenic plants overexpressing GS also exhibit en-
hanced photosynthetic and photorespiration capacities
[21,44],enhanced tolerance to water stress (El-Khatib et al.,
unpublished),and enhanced resistance to phosphinothricine,
a broad-spectrum herbicide that inactivates GS .These
reports provide evidence that enhanced GS expression is
correlated with resistance to different types of stress.More-
over,a higher capacity to accumulate bark storage proteins
has been observed in the GS-overexpressing poplars,which
could be a consequence of their altered nitrogen partitioning,
and contribute to enhanced vegetative growth following dor-
mancy (Jing et al.,unpublished data).These results suggest
that enhanced vegetative growth and development of agricul-
turally important species can be achieved by establishing
lines with enhanced cytosolic GS expression in photosyn-
thetic tissues.The coincidence of GS1 genes with QTLs for
yield  supports this strategy.
This evidence supports a strong role for GS1 in plant
development and crop yield.However,if we consider that the
ectopic GS1 expression in transgenic angiosperms may ac-
celerate the plant life cycle by enhancing vegetative growth
resulting in premature ﬂowering and senescence,the ques-
tion on why angiosperms species evolved without GS1 ex-
pression in mature photosynthetic cells remains unsolved.It
may be speculated that high levels of expression of GS could
result in poor adaptation of plants to their environments.It is
interesting to note that,based on nucleotide sequence analy-
sis,plant GS2 appears to have evolved froma duplicated GS1
gene around the time of land plant evolution ;furthermore,
the ﬁrst multicellular plants evolved with atmospheric oxy-
gen levels similar to present day levels.Transgenics overpro-
ducing GS1 have enhanced photosynthetic and photorespira-
tory capacities [21,44],as also reported for enhanced
expression of GS2 in transgenic tobacco .Taking all this
into consideration,it is tempting to speculate that the sup-
pression of GS1 expression in photosynthetic cells and its
substitution by GS2 occurred when land plants were already
exposed to the present oxygen levels,perhaps as an adaptive
mechanism to overcome the high levels of ammonium re-
leased during photorespiration.Therefore,the role of GS1 in
the development and biomass production could be consid-
ered from a human perspective of plant productivity and
could have a minor relevance,if we take into consideration
the available data for plant land evolution.More data from
other species transformed with GS genes will be necessary to
get a better understanding of the biological relevance of GS1.
3.2.Modiﬁcation of phenylalanine metabolism
Phenylalanine (tyrosine) ammonia-lyase (PAL) catalyzes
the deamination of phenylalanine and tyrosine to cinnamic
and p-coumaric acids,respectively.This is a crucial meta-
bolic step connecting primary nitrogen metabolism through
the shikimate pathway,with the allocation of carbon for the
biosynthesis of phenylpropanoids.Most metabolic ﬂux
through this pathway leads to the biosynthesis of lignin,an
important constituent of wood.As previously reported in
annual plants,PAL in trees is encoded by a multigene family
,but the speciﬁc physiological and biochemical roles of
individual gene members still remain unknown.To our
knowledge,no attempts have been made to manipulate PAL
in trees using genetic engineering approaches.However,
transgenic tobacco plants with altered lignin contents have
been obtained using the bean PAL2 gene in either the sense or
antisense orientation [16,33].These data reinforce a pro-
posed role of PAL as a rate-limiting step in the phenylpro-
panoid pathway.However,recently,it has been shown that
the availability of phenylalanine may limit carbon allocation
to lignin biosynthesis in Pinus taeda .These results sug-
Analysis of growth in 22 independent transgenic lines of poplar (P.
tremula ×P.alba) expressing cytosolic GS1a from Pinus sylvestris.Data
correspond to 2-month-old plants grown in greenhouse.Average data of net
growth and number of leaves of each line are represented with the standard
error of the mean.
590 F.Gallardo et al./Plant Physiology and Biochemistry 41 (2003) 587–594
gest the existence of upstreamcontrol points in the regulation
of reaction catalyzed by PAL.It is,therefore,possible that
primary nitrogen assimilation,or reassimilation of ammo-
nium,is involved in providing phenylalanine for lignin bio-
synthesis.This assumption is supported by the report of
active nitrogen recycling in ligniﬁyng pine cells via the
glutamine synthetase/glutamate synthase (GS/GOGAT)
pathway .It would be interesting to determine if in-
creased growth observed in transgenic poplar overexpressing
GS  is related to a higher capacity for recycling of
ammoniumreleased during lignin biosynthesis.
3.3.Genetic manipulation of the metabolism of polyamines
Alteration of glutamine/glutamate biosynthesis may have
a direct effect on polyamine metabolism.Polyamines are
nitrogen-rich polycations of lowmolecular weight.They are
involved in a variety of cellular and developmental processes
in plants and have been directly associated with plant stress
responses [8,60].The ﬁrst step in polyamine metabolism is
the biosynthesis of putrescine,which can be accomplished
by two alternative pathways:either from ornithine via the
reaction catalyzed by ornithine decarboxylase (ODC),or
from arginine via the reaction catalyzed by arginine decar-
boxylase (ADC).Since arginine is produced fromornithine,
and ornithine can be released from the catabolic breakdown
of arginine,the relative signiﬁcance of these two pathways,
which leads to the production of putrescine,is unclear.
Modiﬁcation of polyamine levels in plants has been
achieved in transgenic plants by an overproduction of heter-
ologous ADC or ODC enzymes [4,6,7,39,52].Transformed
tobacco overexpressing ADC showed increased levels of
putrescine,leading to signiﬁcant changes in phenotype,in-
cluding altered patterns of plant development .In rice,
enhanced growth under saline stress conditions in transgenic
lines overexpressing ADC was observed .These results
indicate that enhanced polyamine production as a result of
overexpression of ADCmay enhance plant development and
Modiﬁcation of polyamine metabolismhas recently been
reported in poplar cell cultures by the expression of a mouse
ODC cDNA [6,7] (Table 1).Transformed cells showed el-
evated ODCenzyme activities and accumulated higher levels
of putrescine .The activity of endogenous ADC was
unaffected.Interestingly,the inhibition of GS by methionine
sulfoximine led to a substantial reduction of polyamine bio-
synthesis,both in transgenic and control cells suggesting that
ornithine biosynthesis occurs mainly via ammoniumassimi-
lation into glutamine and not from catabolic utilization of
arginine.In grapevine cell suspensions,a similar experimen-
tal model,an increased ammoniumavailability in the culture
mediumresulted in increased levels of putrescine.It has been
proposed that polyamine biosynthesis could be a mechanism
for ammonium detoxiﬁcation under stress conditions .
Furthermore,increased putrescine catabolism has also been
demonstrated in ODC transformed poplar cells .In addi-
tion to resulting in enhanced stress resistance,this may also
result in metabolic intermediates with important functions in
plant growth and development.Interestingly,putrescine is
reported to play a role in the rooting of poplar shoots ,
and in enhancing the frequency of somatic embryogenesis in
cell lines of carrot that overexpress ODC .
Taking into consideration the varied roles ascribed to
polyamines ,metabolic studies of transgenic poplar cells
are extremely valuable for a better understanding of overall
nitrogen metabolism.The generation of transgenic trees with
altered expression of key enzymes of polyamine biosynthesis
will complement these studies and provide newinsights into
relevant issues in forest biotechnology,including rooting,
somatic embryogenesis,and tolerance to abiotic stress.
3.4.Genetic manipulation of glutathione biosynthesis
Sulfur is an essential constituent of all living organisms.In
plants,sulfur is contained in amino acids,in the redox iron-
sulfur centers of some proteins,in sulfolipids,and in a variety
of secondary metabolites.The mechanisms for sulfate uptake
and reduction to sulﬁde are similar to nitrogen uptake and
reduction to ammonium.Once sulfur is reduced to sulﬁde,it
is incorporated into cysteine,the precursor of all other sulfur-
containing molecules in plants,including the tripeptide glu-
tathione .Glutathione (GSH) is a stable thiol compound
involved in redox regulation.In glutathione,cysteine is
linked by peptide bonds to the c-carboxyl group of glutamate
and to the a-amino group of glycine.In plants,glutathione is
present in all subcellular compartments,where it is essential
in protection against oxidative stress and the detoxiﬁcation of
toxins,xenobiotics and heavy metals .
The biosynthesis of GSH has been engineered in trees by
an overexpressing of bacterial genes coding for enzymes
involved in GSH biosynthesis (Table 1) [19,42].In the ﬁrst
step of this metabolic pathway,the enzyme c-glutamyl-
cysteine synthetase (c-ECS) catalyzes the biosynthesis of
c-glutamylcysteine from cysteine and glutamate.In the sec-
ond step,the formation of the tripeptide is catalyzed by the
enzyme glutathione synthetase (GSHs).The overexpression
of GSHs in transgenic trees had no effect in GSH metabo-
lism,however,the overexpression of c-ECS in hybrid poplar
(P.tremula ×P.alba) increased GSH levels in leaves and
roots,when compared to untransformed controls [31,43,54].
These results suggest that the control of GSHbiosynthesis is
exerted at the level of the ﬁrst enzyme committed to the
pathway,representing a key point of connection between
nitrogen and sulfur metabolism.
Recently,there has been an increased interest in the use of
forest trees,particularly Populus for phytoremediation of
environmentally troublesome compounds .Trees are
considered more suitable for phytoremediation than herba-
ceous plants,because of their signiﬁcant biomass and long
life cycles.Thus,transgenic poplars overexpressing c-ECS
have been proposed as tools for phytoremediation of heavy
metals and herbicides.Increased levels of GSHin transgenic
lines of poplar support stimulation of the biosynthesis of
phytochelatins,small polypeptides synthesized from glu-
591F.Gallardo et al./Plant Physiology and Biochemistry 41 (2003) 587–594
tathione that showenhanced capacity for heavy metal detoxi-
ﬁcation .Herbicide resistance is due to the enhanced
capacity of c-ECS transgenics to inactivate pesticides by
conjugation and by transfer of toxin to the vacuole .
4.Potential beneﬁts in forest biotechnology and future
As discussed above,the manipulation of nitrogen metabo-
lismin trees may have a potential impact on forestry produc-
tion.However,most of the studies have been performed in
controlled laboratory conditions or in greenhouses.There-
fore,ﬁeld trials are required to conﬁrm that the laboratory
and greenhouse performance of transgenic plants is main-
tained under ﬁeld conditions.
In the case of genetically engineered trees,ﬁeld tests are
essential to improve our knowledge on the stability of expres-
sion of inserted genes in long-lived species.For commercial
applications,it is critical that transgenes continue to be ex-
pressed throughout the rotation period,which can range from
several years to decades.Field studies that use transgenic
poplar trees overexpressing GS have been initiated at loca-
tions in Spain and in the United States (Fig.3).The ﬁeld sites
have been intensively prepared,including mechanical tilling,
fertilizer application,and periodic elimination of weeds.No
major incidents of insect,pest or disease problems were
noted in either transgenics or controls.No unusual growth
characteristics,such as high incidents of branching,were
observed.Signiﬁcantly,expression of the transgene appeared
to be stable during the two-ﬁrst growing seasons,and in-
creased growth in plant height and stem diameter of trans-
genics trees have been recorded,conﬁrming results of the
greenhouse studies (Jing et al.,unpublished).
Very recently,agronomic and pulping performance of
transgenic trees with altered lignin metabolismwas reported
from a ﬁeld study carried out in two sites,in France and
England .The expression of the transgenes was found to
be stable for the duration of the 4 years study.Plants re-
mained healthy throughout the study,and growth parameters
did not differ from untransformed trees.Interactions with
insects were normal,and no changes in soil microbial com-
munities were detected beneath the transgenic trees.These
results agreed quite well with the data reported by Kaldorf et
al.,which indicated no signiﬁcant differences in the
degree of mycorrhizal colonization between transgenic as-
pen trees (P.tremula ×P.tremuloides) expressing the rol C
gene and non-transgenic controls.These studies were of
special relevance for genetically engineered forest plants,
which have to develop over decades without external fertili-
In principle,the use of transgenic trees in plantation for-
estry may present fewer concerns for consumers than trans-
genic food crops plants,because the ﬁnal consumer products
are not ingested and,therefore,no effect on the human health
is expected.As occurs with transgenic herbaceous plants,
another important issue to be considered for the implemen-
tation of transgenic forest crops is the assessment of environ-
mental risks associated with the spread of transgenes to
native populations.These concerns could be addressed by
co-engineering reproductive sterility in transgenic lines .
This paper is dedicated to Professor Pierre Gadal on the
occasion of his 65th birthday.F.G.and F.M.C.are specially
Perspectives on transgenic tree research.The picture at the top
corresponds to in vitro-cultivated transgenic poplar (P.tremula ×P.alba)
overexpressing GS1 .Higher growth of transgenic poplars,with respect
to control (bottle on the right) plants,was observed in vitro and in green-
house studies [20,22].Approved ﬁeld trials are necessary to conﬁrm the
higher growth and development of transgenic trees in natural conditions.
The picture at the bottomcorresponds to transgenics overexpressing cytoso-
lic GS1 in a ﬁeld test in the province of Granada (Spain).The test was started
in 2001 with rooted plants of 50 cm in height.The picture was taken on
October 2002 and the height of the trees was approximately 5 m.
592 F.Gallardo et al./Plant Physiology and Biochemistry 41 (2003) 587–594
indebted to Professor Gadal for providing themthe opportu-
nity to work in his laboratory and for maintaining research
collaboration for many years.
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